Inositol Depletion Induced by Acute Treatment of the Bipolar Disorder Drug Valproate Increases Levels of Phytosphingosine.

Bipolar disorder (BD) is a severe psychiatric illness affecting ∼1% of the world population. Valproate (VPA) and lithium, widely used for the treatment of BD, are not universally effective. These drugs have been shown to cause inositol depletion, but translating this observation to a specific therapeutic mechanism has been difficult, hampering the development of more effective therapies. We have shown previously in yeast that chronic VPA treatment induces the unfolded protein response due to increasing ceramide levels. To gain insight into the mechanisms activated during acute VPA treatment, we performed a genome-wide expression study in yeast treated with VPA for 30 min. We observed increased mRNA and protein levels of RSB1, which encodes an exporter of long chain bases dihydrosphingosine (DHS) and phytosphingosine (PHS), and further saw that VPA increased sensitivity of an rsb1Δ mutant to PHS, suggesting that VPA increases long chain base levels. Consistent with this, PHS levels were elevated in wild type and, to a greater extent, in rsb1Δ cells. Expression of ORM genes (negative regulators of PHS synthesis) and of fatty acid elongase genes FEN1 and SUR4 were decreased, and expression of YOR1 (exporter of PHS-1P) and DPL1 (lyase that degrades DHS-1P and PHS-1P) was increased. These effects were more pronounced in medium lacking inositol, and were mirrored by inositol starvation of an ino1Δ mutant. These findings provide a metabolic explanation as to how VPA-mediated inositol depletion causes increased synthesis of PHS and further support the therapeutic relevance of inositol depletion as a bipolar disorder treatment.

Valproate Induces the Unfolded Protein Response by Increasing Ceramide Levels.

Bipolar disorder (BD), which is characterized by depression and mania, affects 1-2% of the world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproate (VPA) is one of the most widely used drugs for the treatment of BD, but the therapeutic mechanism of action of this drug is not understood. This knowledge gap has hampered the development of effective treatments. To identify candidate pathways affected by VPA, we performed a genome-wide expression analysis in yeast cells grown in the presence or absence of the drug. VPA caused up-regulation of FEN1 and SUR4, encoding fatty acid elongases that catalyze the synthesis of very long chain fatty acids (C24 to C26) required for ceramide synthesis. Interestingly, fen1Δ and sur4Δ mutants exhibited VPA sensitivity. In agreement with increased fatty acid elongase gene expression, VPA increased levels of phytoceramide, especially those containing C24-C26 fatty acids. Consistent with an increase in ceramide, VPA decreased the expression of amino acid transporters, increased the expression of ER chaperones, and activated the unfolded protein response element (UPRE), suggesting that VPA induces the UPR pathway. These effects were rescued by supplementation of inositol and similarly observed in inositol-starved ino1Δ cells. Starvation of ino1Δ cells increased expression of FEN1 and SUR4, increased ceramide levels, decreased expression of nutrient transporters, and induced the UPR. These findings suggest that VPA-mediated inositol depletion induces the UPR by increasing the de novo synthesis of ceramide.

Control of Plasma Membrane Permeability by ABC Transporters.

ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2.

SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes.

Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.

Myristate-derived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties.

BACKGROUND: Myristate is a novel potential substrate for sphingoid base synthesis. RESULTS: Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases. CONCLUSION: d16:0 and d16:1 sphingolipids constitute an appreciable proportion of cardiac dihydrosphingosine and dihydroceramide, with distinct biological roles. SIGNIFICANCE: This pool of sphingolipids may play a heretofore unsuspected role in myocardial pathology or protection. The enzyme serine palmitoyltransferase (SPT) catalyzes the formation of the sphingoid base "backbone" from which all sphingolipids are derived. Previous studies have shown that inhibition of SPT ameliorates pathological cardiac outcomes in models of lipid overload, but the metabolites responsible for these phenotypes remain unidentified. Recent in vitro studies have shown that incorporation of the novel subunit SPTLC3 broadens the substrate specificity of SPT, allowing utilization of myristoyl-coenzyme A (CoA) in addition to its canonical substrate palmitoyl-CoA. However, the relevance of these findings in vivo has yet to be determined. The present study sought to determine whether myristate-derived d16 sphingolipids are represented among myocardial sphingolipids and, if so, whether their function and metabolic routes were distinct from those of palmitate-derived d18 sphingolipids. Data showed that d16:0 sphingoid bases occurred in more than one-third of total dihydrosphingosine and dihydroceramides in myocardium, and a diet high in saturated fat promoted their de novo production. Intriguingly, d16-ceramides demonstrated highly limited N-acyl chain diversity, and in vitro enzyme activity assays showed that these bases were utilized preferentially to canonical bases by CerS1. Functional differences between myristate- and palmitate-derived sphingolipids were observed in that, unlike d18 sphingolipids and SPTLC2, d16 sphingolipids and SPTLC3 did not appear to contribute to myristate-induced autophagy, whereas only d16 sphingolipids promoted cell death and cleavage of poly(ADP-ribose) polymerase in cardiomyocytes. Thus, these results reveal a previously unappreciated component of cardiac sphingolipids with functional differences from canonical sphingolipids.

Haemodialysis machine designation for patients with chronic Hepatitis B virus: A practice and attitudes survey of Australian renal healthcare workers.

BACKGROUND: To reduce the risk of viral transmission, guidelines recommend the use of designated haemodialysis machines and patient isolation for patients with chronic hepatitis B virus (HBV). These practices are without a strong evidence base, and may no longer be necessary in the setting of heat disinfection programs and standard precautions. METHODS: An online cross-sectional survey was developed for renal clinicians across Australia and New Zealand to explore infection prevention policy concerning patients with chronic HBV in haemodialysis units. We sought to determine whether psychosocial and cultural impacts might result from the mandatory use of machine designation and patient isolation practices, as perceived by multidisciplinary healthcare workers with experience working with this patient population. RESULTS: Sixty-seven responses from 27 health districts across all states of Australia and one New Zealand district were received. Most respondents were from urban areas (65%), and were nurses (87%). 50% of health districts reported using designated machines, while 32% isolate patients. Lack of necessary resources limited the use of designated machines (57%), and patient isolation (78%). Respondents not routinely using these precautions were more likely to express concerns regarding patient psychosocial wellbeing and cultural appropriateness. Overall, 30% of respondents expressed concerns regarding the cultural appropriateness of these recommendations. CONCLUSION: We demonstrate wide variation in haemodialysis infection prevention and control policy and practice with regards to managing patients with chronic HBV. While use of standard precautions and machine disinfection are consistently applied, resource availability and concerns for patient psychosocial wellbeing limit adherence to international guidelines.

Acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages.

Periodontal disease is more prevalent and severe in patients with diabetes than in nondiabetic patients. In addition to diabetes, a large number of studies have demonstrated an association between obesity and chronic periodontal disease. However, the underlying mechanisms have not been well understood. Since plasma free fatty acids (FAs) are elevated in obese patients and saturated FAs such as palmitic acid (PA) have been shown to increase host inflammatory response, we sought to find out how PA interacts with lipopolysaccharide (LPS), an important pathological factor involved in periodontal disease, to enhance inflammation. We found that whereas low concentration of LPS (1 ng/ml) stimulated interleukin (IL)-6 expression in RAW 264.7 macrophages, PA further augmented it fourfold. Besides IL-6, PA amplified the stimulatory effect of LPS on a large amount of Toll-like receptor (TLR)4-mediated expression of proinflammatory signaling molecules such as IL-1 receptor-associated kinase-like 2 and proinflammatory molecules, including monocyte chemotactic protein-1 and colony-stimulating factor. We also observed that PA augmented TLR4 but not TLR2 signal, and the augmentation was mediated by nuclear factor-κB (NF-κB) pathways. To further elucidate the regulatory mechanism whereby PA amplifies LPS signal, our studies showed that PA and LPS synergistically increased hydrolysis of sphingomyelin by stimulating acid sphingomyelinase (ASMase) activity, which contributed to a marked increase in ceramide production and IL-6 upregulation. Taken together, this study has demonstrated that PA markedly augments TLR4-mediated proinflammatory signaling triggered by low concentration of LPS in macrophages, and ASMase plays a key role in the augmentation.

Prescribing Stimulants for Children and Adolescents With Attention-Deficit/Hyperactivity Disorder and Co-occurring Cannabis Use: Considerations for Managing a Clinical Dilemma.

Attention-deficit/hyperactivity disorder (ADHD) is highly prevalent in the pediatric population, with 11% of children and adolescents having ever been diagnosed with the disorder.1 The management of ADHD in the setting of co-occurring cannabis use, which is more prevalent in adolescents with ADHD than in the general population, is an increasingly common dilemma facing clinicians, in part due to recent changes in social acceptability, access, usage, and state-level legal status of cannabis.2 Clinicians face several considerations, including the following: the confounding effects of cannabis use on assessment and management of ADHD symptoms; the potential reduction in risk of substance use when ADHD symptoms are well managed; and the increased risk of misuse and diversion of stimulants in patients with ongoing cannabis use.2.

Treatment of Adults with Autism and Major Depressive Disorder Using Transcranial Magnetic Stimulation: An Open Label Pilot Study.

Patients with autism spectrum disorder (ASD) are at high risk for comorbid major depressive disorder (MDD), which can severely impair functioning and quality of life. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique, which is Food and Drug Administration (FDA) cleared for the treatment of MDD in adults. Despite demonstrated efficacy in the treatment of depression, there are limited data on the use of rTMS in patients with ASD and comorbid MDD. We hypothesized that a standard rTMS protocol for MDD would reduce depressive symptoms for adults with ASD and MDD. Secondarily, we investigated whether this treatment would also reduce core ASD symptoms. Participants of 18-65 years old with ASD and MDD without any medication changes in the last month were eligible for this open-label trial. Participants underwent 25 sessions of rTMS (figure-of-eight coil, 100-120% resting motor threshold, 10 Hz, 3,000 pulses per session) applied to the left dorsolateral prefrontal cortex. Thirteen participants enrolled in the study, with two withdrawing due to tolerability, and one excluded from analysis. Overall, side effects were mild and rTMS was well tolerated. The Hamilton rating scale for depression (HAM-D17 ) improved 13.5 points (IQR 5-15), and 40% of participants achieved remission (HAM-D17 ≤ 7) after rTMS treatment. Informant clinical scales of core symptoms of autism also suggested improvement with rTMS, though no change was observed by the participants themselves. Thus, this open-label trial suggests that high-frequency rTMS is well tolerated by adults with autism and MDD, with improvement in depressive symptoms and possible effects on core autism symptoms. Autism Res 2020, 13: 346-351. © 2020 International Society for Autism Research,Wiley Periodicals, Inc. LAY SUMMARY: This study evaluated the safety and effects of repetitive transcranial magnetic stimulation (rTMS) on depression and autism symptoms in individuals with both major depressive disorder and autism spectrum disorder. rTMS was well tolerated by the participants, depression improved with treatment, and family members' assessment of autism symptoms improved as well. This study supports the need for further work to evaluate rTMS in individuals who have both autism and depression.

Effectiveness of a training program for carers to recognize depression among older people.

OBJECTIVE: Depression is a mental disorder that is frequently not detected among older people. The current study was designed to evaluate the effectiveness of a training program to assist carers to better recognize depression among older people in both community and residential care settings. METHODS: In total, 52 professional carers (26 in community care, 26 in residential care) across a range of occupations completed a four session (for personal care attendants) or six session (for registered nurses or managers) training program. The program provided training for staff to identify and respond appropriately to signs of depression. In addition, nurses and managers were trained on the use of screening tools and referral processes. Outcomes were evaluated at post-test, and 6-month follow-up. RESULTS: The results demonstrated that for all groups training was effective in increasing carers' knowledge of depression and self-efficacy in detecting depression, as well as reducing the barriers to care at both post-test and 6-month follow-up. CONCLUSIONS: The training program evaluated in the current study was effective in increasing the level of skills necessary for care staff to better detect depression among older people in both community and residential care settings. Further research is needed to determine if these improved skills are sustained over time, and if they actually improve the level of recognition of depression among older people.

Depression training program for caregivers of elderly care recipients: implementation and qualitative evaluation.

The prevalence of untreated depression is high among older adults who receive care in residential facilities or in their own omes and is associated with reduced quality of life and other medical conditions, Research has suggested a number of rea-p sons for the low detection and treatment rates for this problem, including lack of knowledge and efficacy among those who provide direct care and poor communication between these caregivers and senior staff, and between senior staff and genera practitioners. In this study, we report on the implementation of a training program for care staff that aims to address these issues. Focus groups with participants who completed the training indicated a high level of satisfaction with the program and reported improvements in knowledge, self-efficacy, and communication within services. It is recommended that the program be more systematically evaluated in relation to its longer term effects on care provider practices and the well-being of depressed elderly care recipients.

Prosteatotic genes are associated with unsaturated fat suppression of saturated fat-induced hepatic steatosis in C57BL/6 mice.

Both high sugar and fat diets can induce prosteatotic genes, leading to obesity and obesity-associated diseases, including hepatic steatosis. Unsaturated fat/fatty acid (USFA) reduces high sugar-induced hepatic steatosis by inhibiting the induced prosteatotic genes. In contrast, it is still unclear how USFA ameliorates saturated fat/fatty acid (SFA)-induced hepatic steatosis. As sugar and fat have different transport and metabolic pathways, we hypothesized that USFA suppressed SFA-induced hepatic steatosis via a different set of prosteatotic genes. To test this, we implemented high SFA vs USFA diets and a control diet in C57BL/6 mice for 16 weeks. Severe hepatic steatosis was induced in mice fed the SFA diet. Among a nearly complete set of prosteatotic genes, only the stearoyl-coenzyme a desaturase 1 (Scd1), cluster of differentiation 36 (Cd36), and peroxisome proliferator-activated receptor γ (Pparγ) genes that were differentially expressed in the liver could contribute to SFA-induced steatosis or the alleviative effect of USFA. That is, the SFA diet induced the expression of Cd36 and Pparγ but not Scd1, and the USFA diet suppressed Scd1 expression and the induction of Cd36 and Pparγ. These findings were mainly recapitulated in cultured hepatocytes. The essential roles of SCD1 and CD36 were confirmed by the observation that the suppression of SCD1 and CD36 with small interfering RNA or drug treatment ameliorated SFA-induced lipid accumulation in hepatocytes. We thus concluded that SCD1, CD36, and PPARγ were essential to the suppression of SFA-induced hepatic steatosis by main dietary USFA, which may provide different therapeutic targets for reducing high-fat vs sugar-induced hepatic steatosis.

Sphingolipid regulators of cellular dysfunction in Type 2 diabetes mellitus: a systems overview.

Climbing obesity rates have contributed to worldwide increases in obesity-associated diseases, including the metabolic syndrome and Type 2 diabetes mellitus (T2DM). Sphingolipids, an important class of structural and signaling lipids, have emerged as key players in the development and pathogenesis of insulin resistance and T2DM. More specifically, sphingolipids have been demonstrated to play integral roles in lipotoxicity and other aspects of pathogenesis in T2DM, although the cellular mechanisms by which this occurs and by which sphingolipid metabolism is dysregulated in T2DM remain under investigation. This review summarizes current knowledge of sphingolipid metabolism and signaling in key organs and tissues affected by T2DM, including the pancreas, adipose tissue, skeletal muscle, cardiovascular system and liver, and highlights areas that ripe for future investigation.

The yeast oxysterol binding protein Kes1 maintains sphingolipid levels.

The oxysterol binding protein family are amphitropic proteins that bind oxysterols, sterols, and possibly phosphoinositides, in a conserved binding pocket. The Saccharomyces cerevisiae oxysterol binding protein family member Kes1 (also known as Osh4) also binds phosphoinositides on a distinct surface of the protein from the conserved binding pocket. In this study, we determine that the oxysterol binding protein family member Kes1 is required to maintain the ratio of complex sphingolipids and levels of ceramide, sphingosine-phosphate and sphingosine. This inability to maintain normal sphingolipid homeostasis resulted in misdistribution of Pma1, a protein that requires normal sphingolipid synthesis to occur to partition into membrane rafts at the Golgi for its trafficking to the plasma membrane.

Ceramide synthase 5 mediates lipid-induced autophagy and hypertrophy in cardiomyocytes.

Diabetic cardiomyopathy (DbCM), which consists of cardiac hypertrophy and failure in the absence of traditional risk factors, is a major contributor to increased heart failure risk in type 2 diabetes patients. In rodent models of DbCM, cardiac hypertrophy and dysfunction have been shown to depend upon saturated fatty acid (SFA) oversupply and de novo sphingolipid synthesis. However, it is not known whether these effects are mediated by bulk SFAs and sphingolipids or by individual lipid species. In this report, we demonstrate that a diet high in SFA induced cardiac hypertrophy, left ventricular systolic and diastolic dysfunction, and autophagy in mice. Furthermore, treatment with the SFA myristate, but not palmitate, induced hypertrophy and autophagy in adult primary cardiomyocytes. De novo sphingolipid synthesis was required for induction of all pathological features observed both in vitro and in vivo, and autophagy was required for induction of hypertrophy in vitro. Finally, we implicated a specific ceramide N-acyl chain length in this process and demonstrated a requirement for (dihydro)ceramide synthase 5 in cardiomyocyte autophagy and myristate-mediated hypertrophy. Thus, this report reveals a requirement for a specific sphingolipid metabolic route and dietary SFAs in the molecular pathogenesis of lipotoxic cardiomyopathy and hypertrophy.